Bottom Line:
Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe.Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3.Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.

ABSTRACTMultiple KH-domain proteins, collectively known as vigilins, are evolutionarily highly conserved proteins that are present in eukaryotic organisms from yeast to metazoa. Proposed roles for vigilins include chromosome segregation, messenger RNA (mRNA) metabolism, translation and tRNA transport. As a step toward understanding its biological function, we have identified the fission yeast vigilin, designated Vgl1, and have investigated its role in cellular response to environmental stress. Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe. Instead, Vgl1 is required for cell survival under thermal stress, and vgl1Δ mutants lose their viability more rapidly than wild-type cells when incubated at high temperature. As for Scp160 in S. cerevisiae, Vgl1 bound polysomes accumulated at endoplasmic reticulum (ER) but in a microtubule-independent manner. Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3. Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.

Figure 4: Assembly and disassembly of heat shock-induced Vgl1 granules. (A) Vgl1-GFP was visualized after growth to mid-logarithmic phase at 30°C (time 0) or after a shift to 42°C for the time indicated. Bar: 5 µm. (B) Whole-cell protein extracts from cells expressing Vgl1-TAP incubated at 42°C for the times indicated were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS–PAGE and subjected to immunoblotting using anti-PAP (Peroxidase-Anti-Peroxidase soluble complex) antibodies to reveal Vgl1 proteins. Antibodies against α-tubulin were used as controls. The relative level of Vgl1 is indicated beneath each lane (average of two independent experiments). (C) Localization of Vgl1-GFP after a 15-min incubation at 42°C (time 0) followed by a shift back to 30°C for the times indicated. (D) The indicated strains were grown in liquid culture to mid-logarithmic phase at 30°C and shifted to 45°C. Samples of 500 cells taken at the indicated times after the shift to 45°C were plated in duplicate onto YES agar and incubated at 30°C. After 3 days of growth, viability was scored as a percentage of the number of colonies formed by the sample taken at time zero.

Mentions:
Intriguingly, during the characterization of Vgl1 in S. pombe, we found that under thermal stress, Vgl1 is rapidly relocalized to cytoplasmic granules without changes of ER structures (Figure 2B). To investigate the dynamic distribution of Vgl1, again we monitored the Vgl1-containing complexes using sucrose gradient fractionation. In line with the relocalization of Vgl1, we found that disruption of the polysomes by the treatment of cells under thermal stress shifted the Vgl1 signal to the top of the gradient (Figure 3B). Consistent with a function in RNA metabolism, these Vgl1 granules were overlapping with SYTO nucleic acid stains specific for RNA (Figure 2E), suggesting that Vgl1 might escort RNA from ER-associated polyribosomes to the cytosol under thermal stress. Detailed analysis revealed that this process is highly dynamic and reversible. Upon thermal stress, the accumulation of Vgl1 at the ER rapidly disappeared within 2 min and small patches of granule-like structures occurred in the cytoplasm at around 5 min (Figure 4A). By 10 min these granule structures become evident and remain stable for up to 60 min. Western blotting demonstrated that these changes in cellular localization occurred in the absence of any effect on Vgl1 protein level (Figure 4B). Following removal of the thermal stress, these granule-like structures rapidly dispersed, and Vgl1 accumulated again at the ER (Figure 4C). In line with these data, we found that vgl1Δ mutants were more susceptible to thermal stress and, when incubated at high temperature, lost their viability more rapidly than wild-type cells (Figure 4D). We conclude that Vgl1 mediates cell survival under thermal stress.Figure 4.

Figure 4: Assembly and disassembly of heat shock-induced Vgl1 granules. (A) Vgl1-GFP was visualized after growth to mid-logarithmic phase at 30°C (time 0) or after a shift to 42°C for the time indicated. Bar: 5 µm. (B) Whole-cell protein extracts from cells expressing Vgl1-TAP incubated at 42°C for the times indicated were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS–PAGE and subjected to immunoblotting using anti-PAP (Peroxidase-Anti-Peroxidase soluble complex) antibodies to reveal Vgl1 proteins. Antibodies against α-tubulin were used as controls. The relative level of Vgl1 is indicated beneath each lane (average of two independent experiments). (C) Localization of Vgl1-GFP after a 15-min incubation at 42°C (time 0) followed by a shift back to 30°C for the times indicated. (D) The indicated strains were grown in liquid culture to mid-logarithmic phase at 30°C and shifted to 45°C. Samples of 500 cells taken at the indicated times after the shift to 45°C were plated in duplicate onto YES agar and incubated at 30°C. After 3 days of growth, viability was scored as a percentage of the number of colonies formed by the sample taken at time zero.

Mentions:
Intriguingly, during the characterization of Vgl1 in S. pombe, we found that under thermal stress, Vgl1 is rapidly relocalized to cytoplasmic granules without changes of ER structures (Figure 2B). To investigate the dynamic distribution of Vgl1, again we monitored the Vgl1-containing complexes using sucrose gradient fractionation. In line with the relocalization of Vgl1, we found that disruption of the polysomes by the treatment of cells under thermal stress shifted the Vgl1 signal to the top of the gradient (Figure 3B). Consistent with a function in RNA metabolism, these Vgl1 granules were overlapping with SYTO nucleic acid stains specific for RNA (Figure 2E), suggesting that Vgl1 might escort RNA from ER-associated polyribosomes to the cytosol under thermal stress. Detailed analysis revealed that this process is highly dynamic and reversible. Upon thermal stress, the accumulation of Vgl1 at the ER rapidly disappeared within 2 min and small patches of granule-like structures occurred in the cytoplasm at around 5 min (Figure 4A). By 10 min these granule structures become evident and remain stable for up to 60 min. Western blotting demonstrated that these changes in cellular localization occurred in the absence of any effect on Vgl1 protein level (Figure 4B). Following removal of the thermal stress, these granule-like structures rapidly dispersed, and Vgl1 accumulated again at the ER (Figure 4C). In line with these data, we found that vgl1Δ mutants were more susceptible to thermal stress and, when incubated at high temperature, lost their viability more rapidly than wild-type cells (Figure 4D). We conclude that Vgl1 mediates cell survival under thermal stress.Figure 4.

Bottom Line:
Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe.Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3.Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.

ABSTRACTMultiple KH-domain proteins, collectively known as vigilins, are evolutionarily highly conserved proteins that are present in eukaryotic organisms from yeast to metazoa. Proposed roles for vigilins include chromosome segregation, messenger RNA (mRNA) metabolism, translation and tRNA transport. As a step toward understanding its biological function, we have identified the fission yeast vigilin, designated Vgl1, and have investigated its role in cellular response to environmental stress. Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe. Instead, Vgl1 is required for cell survival under thermal stress, and vgl1Δ mutants lose their viability more rapidly than wild-type cells when incubated at high temperature. As for Scp160 in S. cerevisiae, Vgl1 bound polysomes accumulated at endoplasmic reticulum (ER) but in a microtubule-independent manner. Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3. Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.